Compositions and methods for improved cell-based botulinum neurotoxin assays

ABSTRACT

Methods for a cell-based assay for Botulinum neurotoxin are provided in which a transfected cell that produces a reporting peptide is contacted with a Botulinum neurotoxin in media having a sub-physiological osmolarity and a temperature that is above physiological temperature. This combination provides an unexpected synergistic effect in reducing the EC50 of the cell-based assay relative to an analogous cell-based assay performed at physiological osmolarity and temperature.

This application is a continuation of United States Patent ApplicationPublication No. 2017/0,097,350, filed Dec. 19, 2016, which is acontinuation of U.S. Pat. No. 9,526,345, filed Aug. 8, 2014, whichclaims the benefit of priority to U.S. Provisional Application No.61/864,436, filed on Aug. 9, 2013. These and all other extrinsicmaterials discussed herein are incorporated by reference in theirentirety. Where a definition or use of a term in an incorporatedreference is inconsistent or contrary to the definition of that termprovided herein, the definition of that term provided herein applies andthe definition of that term in the reference does not apply.

FIELD OF THE INVENTION

The field of the invention is protease assays related to botulinumtoxins.

BACKGROUND

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum, andare among the most potent toxins known. These toxins are awell-recognized source of food poisoning, often resulting in seriousharm or even death of the victims. There are seven structurally relatedbotulinum neurotoxins or serotypes (BoNT/A-G), each of which is composedof a heavy chain (˜100 KD) and a light chain (˜50 KD). The heavy chainmediates toxin entry into a target cell through receptor-mediatedendocytosis. Once internalized, the light chain is translocated from theendosomal vesicle lumen into the cytosol, and acts as a zinc-dependentprotease to cleave proteins that mediate vesicle-target membrane fusion(“substrate proteins”).

These BoNT substrate proteins include plasma membrane protein syntaxin,peripheral membrane protein SNAP-25, and a vesicle membrane proteinsynaptobrevin (Syb). These proteins are collectively referred to as theSNARE (soluble N-ethylmaleimide-sensitive factor attachment proteinreceptor) proteins. Cleavage of SNARE proteins blocks vesicle fusionwith plasma membrane and abolishes neurotransmitter release atneuromuscular junction. Among the SNARE proteins, syntaxin and SNAP-25usually reside on the target membrane and are thus referred to ast-SNAREs, while synaptobrevin is found exclusively with synapticvesicles within the synapse and is called v-SNARE. Together, these threeproteins form a complex that is thought to be the minimal machinery tomediate the fusion between vesicle membrane and plasma membrane. BoNT/A,E, and C cleave SNAP-25, BoNT/B, D, F, G cleave synaptobrevin (Syb), atsingle but different sites. BoNT/C also cleaves syntaxin in addition toSNAP-25.

Due to their threat as a source of food poisoning, and as bioterrorismweapons, there is a need to sensitively and speedily detect BoNTs.Currently, the most sensitive method to detect toxins is to performtoxicity assay in mice. Such methods, however, entail considerableexpense and are subject to regulations related to animal testing.

As a result, there is a growing interest in developing alternatives toanimal-based methods for BoNT characterization. An attractivealternative is the use of cell-based assays, which maintain thereceptor-based internalization and subsequent cleavage of the BoNTmolecule that is generally absent from conventional in vitro assays.Such cell-based assays utilize cells that express constructs that areresponsive to the BoNT, in some instances utilizing Förster resonanceenergy transfer (FRET) and in other instances utilizing non-FRET methodsto provide fluorescence useful for the detection and characterization ofBoNTs. Examples can be found in United States Patent Application No.2004/0,191,887 (to Chapman), United States Patent Application No.2006/0,134,722 (to Chapman), U.S. Pat. No. 7,208,285 (to Steward), U.S.Pat. No. 7,183,066 (to Fernandez-Salas), and United States PatentApplication No. 2011/0,033,866 (to Atapattu), each of which isincorporated herein by reference in their entirety. For someapplications, however, the sensitivity of such cell-based methods can belacking. For example, United States Patent Application No.2006/0,134,722 (to Chapman) discloses that EC50 value of cell based FRETassay to detect BoNTs is in the ≥10 pM range.

International Patent Application No. WO 2014/060373 (to Eisele) reportedenhancement of the sensitivity of cells to intoxication with botulinumtoxin by allowing certain tumor cells that had been primed fordifferentiation into neuronal cells to differentiate in a low osmolaritydifferentiation media for several days to several weeks prior toexposure to the toxin. Sensitivity was determined by lysis of thetreated cells followed by a Western blot method directed towardsSNAP-25. The utility of Western blotting as a quantitative method isconsidered debatable, however, and no data demonstrating the statisticalsignificance of the reported differences was provided.

Although some success has been demonstrated in applying FRET assays todetection of BoNTs, the sensitivity of FRET assay to BoNTs has beenstill undesirable for many purposes. As few as 40 nanograms of BoNT is alethal dose for most people, and samples suspected to contain BoNTs areoften are prior to application to the test process. It is, therefore,strongly desirable to have methods that detect low concentrations ofBoNT.

SUMMARY OF THE INVENTION

Methods and compositions are disclosed that provide for increasedsensitivity in cell-based assays for botulinum toxins. Cells areprovided that express a reporting construct that is responsive tobotulinum toxin. Media compositions are identified that provideincreased sensitivity of the transfected cell response to botulinumtoxin relative to conventional media compositions. In particular, mediacompositions having a reduced sodium concentration relative toconventional cell culture media are contemplated.

One embodiment of the inventive concept is a method of increasing thesensitivity of cell-based detection of a botulinum toxin by providing,in a first media with a sodium concentration greater than 65 mM, atransfected cell that expresses a hybrid protein, where the hybridprotein includes a reporter-containing portion (for example, at or neara terminus of the hybrid protein) and a cleavage site, where thecleavage site is cleaved by a botulinum toxin to release thereporter-containing portion (for example, a portion containing afluorophore) from the remainder of the hybrid protein. The cell is thentransferred to a second media having a sodium concentration of less than50 mM (for example, less than 45 mM) and is contacted with a botulinumtoxin in the second media. In some embodiments the transfected cell isexposed to the botulinum toxin upon transfer to the second media (i.e.without pre-incubation in the second media). Thereafter a signal isobtained from the reporter-containing portion of the hybrid protein. Insuch a method the sensitivity of the transfected cell to botulinum toxincan be increased by a factor of 10 or more relative to a method wherethe cell is maintained in and exposed to botulinum toxin in the firstmedia (i.e. a media with a sodium concentration of at least 65 mM).

In some embodiments the sodium concentration in the second media can bereduced by a reduction in the sodium content of a neurobasal media, forexample by reducing the concentration of sodium chloride and/or sodiumbicarbonate. In some embodiments the first media, the second media, orboth can be of physiological osmotic strength. In other embodiments, thefirst media, the second media, or both can have an osmotic strength thatis less than that of physiological osmotic strength, for example 250mOsm or less.

In some embodiments of the inventive concept a transfected cell asdescribed above is exposed to an elevated (i.e. in excess of 37° C.)temperature during exposure to a botulinum toxin in a sensitizing cellculture media (e.g. cell culture media having a reduced sodium content).Such elevated temperatures can range from 38.0° C. to 41.0° C., and insome embodiments can range from 38.5° C. and 39.5° C. In still otherembodiments a transfected cell as described above can also be exposed tosuch elevated temperatures prior to exposure to a botulinum toxin in asensitizing cell culture media.

Another embodiment of the inventive concept is a method of increasingsensitivity of a cell based assay for Botulinum neurotoxin by providing,in a media having a sub-physiological osmolarity (e.g. 250 mOsm) and atemperature of at least 39° C., a transfected cell that includes areporting peptide cleavable by Botulinum neurotoxin. The cell is broughtinto contact with Botulinum neurotoxin and a cleavage product derivedfrom the reporting peptide is measured. A synergistic decrease in EC50(for example, by a factor of three) for Botulinum neurotoxin is providedrelative to the cell-based assay performed at physiological temperatureand physiological osmolarity. The reporting peptide can include aterminus having a reporter-containing portion that exhibits a signal anda cleavage site that interacts with the Botulinum neurotoxin to producesa cleavage of the reporter-containing portion from a remainder of thereporting peptide. In such an embodiment measuring is performed bymonitoring changes in the signal.

Still another embodiment of the inventive concept is a kit for improvingthe sensitivity of a cell-based assay for a botulinum toxin, where thekit includes a set of standards containing botulinum toxin supplied attwo or more different concentrations in sensitivity enhancing media asdescribed above. Such a kit can include two or more preparationscontaining non-zero concentrations of botulinum toxin in such media, andin some embodiments can include a blank or zero (i.e. media-only)preparation. Such a kit can be used to prepare and/or verifydose/response curves useful for quantitation of botulinum toxin in asample (for example, in the preparation of a calibration curve orverification of assay performance relative to a calibration curve storedin memory).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C show the response of transfected cells to botulinumtoxin and fragments thereof at different temperatures. FIG. 1A shows theresponse of transfected cells to botulinum holotoxin at differenttemperatures. FIG. 1B shows the response of transfected cells tobotulinum holotoxin or to the light chain of botulinum toxin atdifferent temperatures. FIG. 1C shows the response of transfected cellsto botulinum holotoxin in the presence of the heavy chain of botulinumtoxin at different temperatures.

FIG. 2 shows the response of transfected cells to botulinum toxin atdifferent pre-toxin exposure (i.e. culture) temperatures and toxinexposure (i.e. assay) temperatures.

FIGS. 3A and 3B show the effect of media ionic strength on the responseof transfected cells to botulinum toxins at different temperatures. FIG.3A shows the response of transfected cells in a media having an ionicstrength of 270 mM. FIG. 3B shows the response of transfected cells toBotulinum toxin in a media having an ionic strength of 250 mM.

FIGS. 4A and 4B show the effects of elevated temperatures on theresponse of transfected cells to botulinum toxin and in the absence ofbotulinum toxin. FIG. 4A shows the response of transfected cells tobotulinum toxin at temperatures up to 41° C. FIG. 4B shows brightfieldand fluorescence photomicrographs of the response of transfected cellsto temperatures of up to 41° C. in the absence of botulinum toxin.

FIG. 5 shows the effect of increased temperature on a cell-based assayfor BoNT/E toxin.

FIGS. 6A and 6B show the effect of media with reduced sodiumconcentration on cell-based assays for botulinum toxin. FIG. 6A showsthe effect of the use of proprietary media with different concentrationsof added NaCl. FIG. 6B shows photomicrographs of transfected cellsexposed to custom culture media containing different concentrations ofNaCl.

FIG. 7 shows the response of transfected cells to botulinum toxin inmedia with different sodium concentrations and at different time pointsfollowing exposure to the toxin.

FIGS. 8A, 8B, and 8C show the result of exposure of transfected cells tobotulinum toxin in media with reduced sodium content for differentlengths of time prior to the restoration of normal sodiumconcentrations. FIG. 8A shows the effects when transfected cell arecultured in conventional media prior to exposure to botulinum toxin.FIG. 8B shows the effects when the transfected cells are cultured in lowsodium content media prior to exposure to botulinum toxin.

FIGS. 9A and 9B show the result of reducing sodium bicarbonateconcentration in media used in cell-based assays for botulinum toxin.FIG. 8A shows dose/response curves in media with different concentrationof sodium bicarbonate. FIG. 8B shows the effect on EC50s calculated fromsuch dose/response curves as a function of sodium bicarbonateconcentration in the media. FIG. 8C shows the effect of pre-incubationwith either conventional media or media with reduced sodium contentprior to the performance of a cell-based assay for botulinum toxin inlow sodium content media.

FIG. 10 shows the effect of replacing sodium with potassium in custommedia used in cell-based assays for botulinum toxin, and in thereduction of sodium and potassium concentrations in those media.

FIG. 11 shows the effect of increasing the osmolarity of low sodiumcontent media used in cell-based assays for botulinum toxin.

FIGS. 12A and 12B show the effects of botulinum toxin fragments andintact botulinum holotoxin on transfected cells in media with reducedsodium content. FIG. 12A shows the effect of adding recombinantbotulinum toxin heavy chain to the transfected cells prior to exposureto the holotoxin. FIG. 12B shows the effect of adding recombinantbotulinum toxin light chains to the transfected cells and the effect ofadding the intact botulinum holotoxin to the transfected cells.

DETAILED DESCRIPTION

The inventive subject matter provides methods in which the sodium ionconcentration of cell culture media utilized in a cell-based botulinumtoxin assay and/or the temperature at which the cell-based botulinumassay is performed is used to provide botulinum assays with enhancedsensitivity. Surprisingly, the inventors have found that reduction ofsodium ion concentration in the cell culture media enhances sensitivityof botulinum toxin in an ion and botulinum toxin-specific manner. Theinventors also identified a narrow range of temperatures over which thesensitivity of such assays, as defined by dose/response curves, isdramatically enhanced.

The inventive subject matter provides methods for improving thesensitivity of cell-based methods for detecting the presence of abotulinum toxin (BoNT). A variety of assays for botulinum toxins thatutilize transfected cells expressing detecting constructs cleavable bythese proteases have been developed. In cell-based assays, specificbinding of the heavy chain of a botulinum toxin by cell surfacereceptors and followed by specific cleavage of a construct that includesa botulinum toxin-specific cleavage site by the light chain of thebotulinum toxin and subsequent release of an indicator moiety (forexample, a fluorescent protein) from the construct provide a high levelof specificity. Prior art methods, however, lacked the sensitivitynecessary for important applications of such assays, for exampleenvironmental testing. This is particularly important considering thepotential use of botulinum toxins as bioweapons.

Unless the context dictates the contrary, all ranges set forth hereinshould be interpreted as being inclusive of their endpoints, andopen-ended ranges should be interpreted to include only commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary.

Cell-based assays require a rigidly controlled environment, utilizingphysiological ion concentrations, osmolarity (i.e. 250-270 mOsm), andtemperatures held at physiological level. Surprisingly, the inventorshave found that reducing the sodium ion concentration of the culturemedia provides a dramatic increase in the sensitivity of some cell-basedBoNT assays. The effect is not seen with potassium ions, is not a resultof changes in osmolarity of the cell culture media, and is not observedwith certain types of botulinum toxins. The inventors have also foundthat elevating the temperature at which the assay is performed by up to4° C. results in a dramatic increase in the sensitivity of such an assaywithout impacting cell viability over the course of the assay.Similarly, decreasing the osmolarity of the cell media also resulted inan increase in sensitivity. These methods can be adopted without theneed for specialized equipment, and the increased sensitivity realizedbroadens the range of applications for these highly specific botulinumtoxin assays.

Methods of the inventive concept provide a transfected cell, which inturn produces a construct or fusion protein. With respect to thetransfected cells expressing the hybrid protein it is generallypreferred that the cell is stably transfected. Nevertheless, transienttransfection is also contemplated. It is still further typicallypreferred that the transfected cell is a neuronal cell. However,numerous other non-neuronal cells (including mammalian cells, insectcells, yeast, bacteria, and artificial cells) are also contemplatedherein. Most typically, the cells will constitutively express the hybridprotein(s) are therefore under appropriate regulatory elements. Inalternative aspects, the expression can also be induced.

Many choices of cell lines are suitable as the host cell for the presentinvention. Preferably, the cell is of a type in which the respectivebotulinum toxin (BoNT) exhibits its toxic activities. In other words,the cells preferably display suitable cell surface receptors, orotherwise allow the toxin to be translocated into the cell sufficientlyefficiently, and allow the toxin to cleave the suitable substratepolypeptide. Specific examples include primary cultured neurons (e.g.,cortical neurons, hippocampal neurons, spinal cord motor neurons, etc);PC12 cells or cell lines derived from PC12 cells; primary culturedchromaffin cells; cultured neuroblastoma cell lines (such as murinecholinergic Neuro2A cell line), human adrenergic SK—N—SH cell lines,NS-26 cell lines, and stem cells (see e.g. Foster and Stringer (1999),Genetic Regulatory Elements Introduced Into Neural Stem and ProgenitorCell Populations, Brain Pathology 9: 547-567). Similarly, neuroendocrineand neuroendocrine-derived cell lines can be used. It should beappreciated, however, that in the instance of recombinant or mutatedBoNTs that are directed towards non-neuronal cell types, that host cellscan be selected from cell lines with the corresponding specificity.

Constructs or fusion proteins of the inventive concept can include areporter-containing portion and a cleavage site. The cleavage site canact as a substrate for the protease activity associated with a botulinumtoxin light chain. Such transfected cells can demonstrate stabletransformation or transient transformation. Cleavage of the cleavagesite releases at least a portion of the reporter-containing portion froma remainder of the construct. The reporter region can include anobservable reporting group or tag, such as a fluorophore which providesan observable fluorescence. Suitable fluorophores include fluorescentdyes, and can include fluorescent proteins such as Green FluorescentProtein (GFP), Cyan Fluorescent Protein (CFP), Yellow FluorescentProtein (YFP), Citrine, Venus, YPet, mStrawberry, and/or mCherryprotein. In some embodiments the hybrid protein can include multiplefluorophores, for example a second fluorophore. Such a secondfluorophore can be located within the reporter region or at a distallocation. For example, the fluorophore of the reporter region (i.e. thefirst fluorophore) can be located proximate to one terminus of thehybrid protein while the second fluorophore can be located proximate toa different terminus of the hybrid protein. Alternatively, both thefirst fluorophore and the second fluorophore may be within the reporterregion. Depending upon the nature of the detection, the firstfluorophore and the second fluorophore can be the same fluorophorespecies, or can be different fluorophore species. For example, in anassay system utilizing FRET detection the first fluorophore and thesecond fluorophore can be different fluorophore species.

In a preferred embodiment of the inventive concept, the reporter regionincludes one or more fluorophores of the same species, which can bearranged so that homo-FRET does not occur to a significant degree (i.e.less than 5% Förster resonance energy transfer). In other embodimentsthe construct can include fluorophores of different species, which canbe arranged so that FRET does not occur to a significant degree (i.e.less than 5% Förster resonance energy transfer). This can beaccomplished, for example, by placing the fluorophores at or neardifferent termini of the construct. In such embodiments the emissionspectra of a first fluorophore can overlap with the excitation spectraof a second fluorophore without significant (i.e. less than 5%) Försterresonance energy transfer, however fluorescence emission of the firstfluorophore is not significantly decreased (i.e. less than 5%) viaquenching and fluorescence emission of the second fluorophore is notsignificantly (i.e. more than 5%) increased via such energy transfer. Inother embodiments of the inventive concept the construct can include afirst fluorophore with an emission spectrum that overlaps the excitationspectrum of a second fluorophore, with position of the fluorophoreswithin the construct arranged such that significant (i.e. >5% Försterresonance energy transfer) occurs between the fluorophores. Fluorescencefrom a construct of the inventive concept can be detected by any meanssuitable for the configuration of the construct, for example includingdirect excitation and emission from each fluorescent species, FRET, andfluorescence anisotropy. In a preferred embodiment, a conventionalmicrowell plate fluorometer configured for direct excitation andemission detection from each fluorophore species can be used.

Green fluorescent protein and its mutations, which fluoresce without theneed for additional cofactors or substrates, are particularly suitablefor use with constructs of the inventive concept. For example, YellowFluorescent Protein (YFP) is a mutation of the Green FluorescentProtein, derived from Aequorea victoria, and has an excitation peak at514 nm and an emission peak at 527 nm. In addition to YFP, it is alsocontemplated to use related Citrine, Venus, and YPet proteins can beused in the reporter-containing portion. These mutations have reducedchloride sensitivity, faster maturation, and increased brightness(product of the extinction coefficient and quantum yield) relative toGFP. Of course, any of the fluorescent proteins mentioned herein can bemodified to include specific characteristics (e.g., spectral) or betruncated to a specific size. It is also contemplated that the reportercontaining portion includes reporters other than fluorescent proteins(e.g., a phosphorescent compound, a luminescent compound, a chromophore,an enzyme, etc.).

In some embodiments of the inventive concept the detection signal ischaracterized prior to exposure of the transfected cells to thebotulinum toxin (BoNT), to provide a baseline signal. This baselinesignal can serve as a basis for comparison to an assay signal obtainedfollowing exposure of the transfected cells to botulinum toxin, and canserve to normalize such an assay signal to at least partially correctfor variations in cell number, density, and/or shape between differenttest sites. For example, the use of a ratio between a post-exposuresignal and the baseline signal can serve to normalize fluorescenceintensity between assays performed in different wells of a microwellplate, thereby reducing the variation between like measurements.Sensitivity can be assessed by preparing a series of such assaysutilizing different concentrations of botulinum toxin to generate adose/response curve, which is typically sigmoidal. Sensitivity can bequantified by determining the concentration of botulinum toxin thatgenerates a response that correlates to a defined portion of the doseresponse curve. For example, a botulinum concentration that correlateswith the midpoint or half-maximal value of the dose/response curve(typically reported as the EC50) can be used as a basis for comparingsensitivity in such assays.

Many different methods can be used to measure sensitivity to botulinumtoxin using a cell-based assay. In one embodiment, an emission ratio ofa first fluorescent protein and a second fluorescent protein that do notform a FRET pair (i.e. demonstrate less than about 5% energy transfervia FRET) can be measured after exposing the transfected cell tobotulinum toxin. In such an embodiment, prior to exposure of the hybridto Botulinum toxin, the construct exhibits a baseline signal, and thefirst fluorescent protein emission and the second fluorescent emissionare separately measured. After exposure to botulinum toxin, thereporter-containing portion comprising the first fluorescent protein iscleaved by the botulinum toxin, and the cleaved reporter-containingportion is subsequently degraded by proteolysis. In such an example theemission intensity of the first fluorescent protein is decreased, whilean emission intensity of the second fluorescent protein remainsessentially the same. The emission measured from this second fluorescentprotein is therefore a function of cell number, density, distribution,and so on, and is not a function of the concentration of botulinumtoxin. As such, the emission from the second fluorescent protein can beused to normalize the emission measured from a fluorophore of thereporter region (in this instance the first fluorescent protein), forexample by using an emission ratio. It should be appreciated that suchan emission ratio is ineffective for data normalization in constructs inwhich the fluorophores are arranged to perform FRET, as the emissionsfrom both fluorophores would change on cleavage of such a construct. Theemission ratio (first fluorescent protein emission/second fluorescentprotein emission) is decreased when the construct interacts withbotulinum toxin. An example of a suitable construct in such anembodiment is one that includes Cyan Fluorescent Protein (CFP) outsideof the reporter region and in which the reporter region includes YellowFluorescent Protein (YFP), configured such that the CFP and YFP do notform a FRET pair. Data related to the degree of YFP degradation (i.e.directly, separately excited YFP emissions and CFP emissions) followingexposure to a botulinum toxin can be collected from a cell expressingsuch a construct. Those emissions can be background subtracted and theYFP emission divided by the CFP emission to control for cell density andreporter expression in the individual cells.

Botulinum toxin responsive emission from a fluorophore of a reporterregion or an emission ratio can be used to generate a dose responsecurve that is useful in quantifying botulinum toxin in a sample and/orto determine sensitivity of an assay to botulinum toxin. Suchsensitivity is frequently expressed as a concentration of the BoNTcorresponding to a characteristic portion of the dose/response curve.For example, a BoNT concentration corresponding to the midpoint of sucha curve is referred to as an EC₅₀.

In one embodiment of the inventive concept, the transfected cells areexposed to the botulinum toxin at a temperature that is elevatedrelative to that at which cell culture and such assays are normallyperformed (i.e. 37.0° C.). It should be appreciated that suchtemperatures are generally considered non-optimal for cell survival, andthat their use is counterintuitive in assays that rely on the use ofviable cells. In a preferred embodiment the temperature at which thetransfected cells are exposed to botulinum toxin is such that thesensitivity is increased at least two-fold (i.e. by a factor of 2)relative to an assay performed at 37.0° C. (i.e. the EC50 of the assayperformed at the elevated temperature is less than half of the EC50 ofthe assay performed at 37.0° C.). Surprisingly, the inventors have foundthat such a sensitivity enhancement occurs within a relatively narrowrange of temperatures. In some embodiments of the inventive concept thetransfected cells are exposed to the botulinum toxin at 38.0° C. to41.0° C. In other embodiments of the inventive concept the transfectedcells are exposed to the botulinum toxin at a temperature between 38.5°C. and 39.5° C.

Alternatively, the transfected cells of the inventive concept can bemaintained at temperatures greater than 37.0° C. prior to exposure tothe botulinum toxin, for example 38.0° C. to 41.0° C. or 38.5° C. and39.5° C. In such embodiments, exposure of the transfected cells tobotulinum toxin can be performed at 37.0° C. Alternatively, in someembodiments transfected cells can be exposed to temperatures greaterthan 37.0° C. (for example, 38.0° C. to 41.0° C. or 38.5° C. and 39.5°C.) both prior to and during exposure to botulinum toxin. In still otherembodiments the temperature of the transfected cells can be rampedduring the performance of the assay. For example, the temperature of thetransfected cells can start at 37.0° C. at the point of introduction ofthe botulinum toxin and then increased (for example, to 41.0° C.) as theassay progresses. Alternatively, in some embodiments the temperature oftransfected cells can start at an elevated temperature (for example,41.0° C.) at the point of introduction of the botulinum toxin and bedecreased to 37.0° C. during the course of the assay.

In embodiments of the inventive concept, a cell-based assay detectingthe presence of botulinum toxin can have an increase of at least twofold in sensitivity to botulinum toxin with a change of conditions(e.g., temperature, osmolarity, extracellular ion concentration, etc.).In one preferred embodiment, the sensitivity to botulinum toxin isincreased at least three fold when the transfected cell is exposed tobotulinum toxin at a higher temperature than 37.0° C., within a range of38° C. to 41° C., or within a range of 38.5° C. to 39.5° C. compared tosensitivity to botulinum toxin at 37.0° C. In another embodiment thesensitivity is increased at least five fold when the transfected cell isexposed to botulinum toxin at a higher temperature than 37.0° C., withina range of 38° C. to 41° C., or within a range of 38.5° C. to 39.5° C.compared to sensitivity to botulinum toxin at 37.0° C. In still otherembodiments the sensitivity is increased at least ten fold when thetransfected cell is exposed to botulinum toxin at a higher temperaturethan 37.0° C., within a range of 38° C. to 41° C., or within a range of38.5° C. to 39.5° C. compared to sensitivity to botulinum toxin at 37.0°C.

It is also thought that reduced osmolarity of a cell media in which thetransfected cell is exposed to BoNT can also enhance sensitivity toBoNT. Without wishing to be bound by theory, the inventors believe thatreduced extracellular osmolarity can result in a modulation of cellularactivity (e.g. neuronal excitability). Especially, in neuronal cells,reduced osmolarity enhances synaptic transmission and neuronalexcitability, which increases the endocytosis rate. Thus, it iscontemplated that a reduction in osmolarity of a cell media in which thetransfected cell is exposed to botulinum toxin, for example to a rangebetween 220 milliOsm and 260 milliOsm may confer an increase insensitivity to botulinum toxin. It should be appreciated that such amodification is counterintuitive, as such conditions can adverselyaffect cell viability. Furthermore, it is also contemplated that reducedosmolarity of the cell media can enhance, for example in a synergisticfashion, an increased sensitivity to botulinum toxin that results from ahigher temperature than 37.0° C.

In another embodiment, increased sensitivity to botulinum toxin can beachieved by decreasing the concentration of specific extracellular ions,for example free (i.e. uncomplexed or non-chelated) calcium. Whenextracellular calcium concentration falls below normal physiologicallevel, the transfected cell can be progressively more excitable. Similarto reduced osmolarity, increased cell excitability can enhanceendocytosis rate and thus enhance the internalization of an appliedbotulinum toxin. Thus, it is also contemplated that reducing calciumconcentration below the physiological level (1.0-1.5 mM) may confer asimilar increase in sensitivity to botulinum toxin. Similarly, additionof calcium chelators (e.g., ethylenediaminetetraacetic acid (EDTA),ethyleneglycoltetraacetic acid (EGTA),1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidtetra(acetoxymethyl) ester (BAPTA/AM), and other organic acids) to thecell media may confer similar increases in sensitivity to botulinumtoxin.

In still another embodiment of the inventive concept, an increase in thesensitivity of a cell-based assay for a botulinum toxin increased whenthe sodium ion concentration of the cell culture media utilized duringthe performance of the assay is reduced. For example, cell culture mediacan be prepared with sodium salts, for example NaCl and/or NaHCO₃,omitted from the formulation. Such cell culture media can have finalsodium ion concentration of less than about 70 mM, about 50 mM, about 40mM, about 30 mM, about 25 mM, or about 20 mM. In a preferred embodimentof the inventive concept the cell culture media used to perform acell-based botulinum toxin assay is less than or equal to about 20 mM.

In performance of a cell-based assay, cells expressing abotulinum-sensitive construct as described above can be pre-incubatedwith a low sodium ion content culture media prior to exposure to thebotulinum toxin, then contacted with a low sodium ion content culturemedia containing botulinum toxin (for example, from an added sample). Inpreferred embodiments of the invention, the cells are not exposed to alow sodium ion content culture medium prior to exposure of the cellsbeyond a brief (i.e. several minute) exchange or wash with low sodiumion content culture media prior to contact with the botulinum toxin.

In some embodiments of the inventive concept sodium ions in the cellculture media can be replaced by other ions that do not show the sodiumion effect (for example, potassium ions) or by other osmolaritymodifying agents (for example, triethylamine N-oxide) to retain thephysiological osmolarity of the cell culture media while still providingthe sensitivity enhancement realized by the reduction in sodium ionconcentration.

It should be appreciated that elevated temperature, reduced mediaosmolarity, reduced extracellular concentration of specific ions (forexample, sodium ions), and additional protein can be combined, and thatsuch combinations can exert a synergistic effect. For example, theinventors have surprisingly found that elevated temperature duringexposure of the transfected cells to botulinum toxin and the use ofmedia with reduced osmolarity has a synergistic effect on theimprovement in sensitivity of a cell based assay.

A number of serotypes of botulinum toxin (BoNT) with different substratespecificities and specific cleavage sites have been identified,including BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/D, BoNT/E, BoNT/F,BoNT/G, and a proposed BoNT/H. In some embodiments of the inventiveconcept, a sensitivity enhancing method can be selective for a specificspecies of BoNT. For example, the use of a low sodium content cellculture medium can result in an enhanced sensitivity for a cell-basedassay for BoNT/A, but have little effect on the sensitivity of acell-based assay BoNT/E. In some embodiments of the inventive conceptthis selective enhancement of the sensitivity to one or more BoNTspecies occurs when the same cell line expressing the same construct isused in characterizing multiple BoNT species.

It is contemplated that a construct of the inventive concept can beresponsive (i.e. act as a substrate) for one or more of such BoNTs.Similarly, it is contemplated that transfected cells expressing hybridreporter/cleavage site bearing proteins that can act as substrates forrecombinant or modified BoNTs with altered specificity and BoNTserotypes and/or isoforms not yet identified will be responsive to themethods of the inventive concept. It is also contemplated thattransfected cells expressing proteins with similar reporter anddifferent cleavage site portions that are responsive to Tetanusneurotoxins (TeNTs) can show similar increases in sensitivity to therespective TeNT when methods of the inventive concept are applied.

In a preferred embodiment, temperatures higher than 37.0° C.significantly and cell culture media with low (i.e. less than about 70mM) sodium ion concentration enhance the sensitivity of the BOCELL™model cell line to botulinum neurotoxin type A (BoNT/A). Such a cellline is described in U.S. Provisional Patent Application No. 61/492,237(filed Jun. 1, 2011) and is incorporated herein. All other extrinsicmaterials discussed herein are similarly incorporated by reference intheir entirety.

BoNTs recognize the cleavage site and cleave the hybrid protein into thereporter-containing portion and the remainder of the hybrid protein. Thecleavage site sequence of the present invention can advantageouslycomprise (a) a SNARE protein, motif, or mutein (or a cleavable portionof these). SNARE proteins are understood to include SNAP-25,synaptobrevin (VAMP), and syntaxin. “Muteins” of a protein should beinterpreted herein as having at least 30% identity with a correspondingnative protein, including for example compositions having at least 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%identity with the native protein. Variations from identity can compriseany or more of additions, deletions and substitutions. Contemplatedmuteins include fragments, truncates and fusion proteins.

Without wishing to be bound by theory, the inventors believe that theobserved increased sensitivity to botulinum toxin at higher temperaturescould be a consequence of increased specific binding and endocytosis ofbotulinum toxin. In the cell-based assay, BoNT must be internalized tothe cell cytoplasm via receptor-mediated endocytosis. It is thereforepossible that anything that causes more BoNT to be internalized, and tointeract with the cleavage site of hybrid proteins/constructs, wouldresult in the transfected cell being more sensitive to BoNT. As shownFIG. 1A, which depicts dose/response curves obtained with botulinumtoxin at different temperatures, relatively small changes in temperatureproduce a surprisingly large effect on the sensitivity of a Botulinumtoxin assay (as determined by EC50). FIG. 1B shows the result of similarstudies performed using intact Botulinum toxin (i.e. holotoxin) and thebotulinum toxin light chain, which has protease activity capable ofcleaving the cell's reporting construct but does not havereceptor-binding activity. The lack of change in the emission ratio ofthe construct noted at conventional and elevated temperatures when thecells are exposed to the light chain indicates that temperature effectsare not a result of generally enhanced endocytosis and are a result ofreceptor-mediated processes. This is supported by the data shown in FIG.1C, which shows the effects of different temperatures on transfectedcells exposed to botulinum toxin in the presence of absence of botulinumtoxin heavy chain, which lacks the ability to cleave the reportingconstruct but can occupy toxin-specific receptor sites. FIG. 1C showsthat the Botulinum toxin heavy chain is effective in blocking theeffects of the holotoxin but less effective at the elevated temperature,indicating that the temperature effect of cell-based assay sensitivitymay be a receptor-mediated process.

Increased expression of botulinum toxin receptor proteins on thetransfected cell's surface may results in enhanced endocytosis ofbotulinum toxin. For example, botulinum toxin A, D and E areinternalized to cell cytoplasm via interaction with synaptic vesicleproteins (SV2) expressed on cell surface. Thus, it is also contemplatedthat co-expression of SV2 protein in the transfected cell may infersimilar increase in sensitivity to botulinum toxin.

Increased activity of endogenous stress response proteins, includingHeat Shock Protein 70 (HSP70) and Heat Shock Protein 90 (HSP90), at thehigher temperature can potentially induce enhanced sensitivity tobotulinum toxin. Both HSP70 and HSP 90 are activated at highertemperatures than physiological temperature range (between 35.0-37.0°C.), and enhance proteolysis activity of the cell. Without wishing to bebound by theory, it is contemplated that increased activity of HSP70 orHSP90 can facilitate breakdown of the reporter-containing portion of thehybrid protein.

Still further, a conformational change of the hybrid protein at thehigher temperature, by which the baseline FRET signal can be augmented,can induce enhanced sensitivity to botulinum toxin. HSP70 functions toaid proper folding of proteins, and increased activity of HSP70 mayinduce a conformational change of the hybrid protein. Therefore, it isalso contemplated that treatment of HSP70 activator (e.g., YM1(2-((Z)-((E)-3-ethyl-5-(3-methylbenzo[d]thiazol-2(3H)-ylidene)-4-oxothiazolidin-2-ylidene)methyl)-1-methylpyridin-1-iumchloride,2-[3-Ethyl-5-(3-methyl-3H-benzothiazol-2-ylidene)-4-oxo-thiazolidin-2-ylidenemethyl]-1-methyl-pyridiniumchloride)) to the transfected cell may infer similar increase insensitivity to botulinum toxin.

It is further contemplated that various conditions described above canbe combined to render further enhanced sensitivity to BoNT in the cellbased assay. For example, reduced osmotic strength and reduced sodiumconcentration in the media can be combined to provide furthersensitivity enhancements. It is contemplated that such combinations canproduce synergistic effects.

Still another embodiment of the inventive concept is a kit that includespreparations that include sensitivity enhancing media as described aboveand one or more botulinum toxins at different concentrations. Forexample, such a kit can include a set of preparations comprising asensitivity enhancing media, wherein each member of the set additionallyincludes a different concentration of botulinum toxin. Such sensitivityenhancing media can be as described above, for example media containingsodium at less than or equal to 50 mM sodium. Such sensitivity enhancingmedia can have physiological or less than physiological osmoticstrength.

In such a kit the preparations can be prepared such that they representa series of botulinum toxin concentrations suitable for use as adose/response curve. Such a dose/response curve can, in turn, be usedfor calibrating a cell-based botulinum toxin assay to produce acharacteristic response. Alternatively, such a kit can contain a limitednumber of such preparations (for example one, two, or threepreparations) that may not be suitable for producing a dose/responsecurve but that can be used to verify that a cell-based botulinum assayis producing results consistent with those of a complete dose/responseor calibration curve that is stored in computer memory. Optionally, sucha kit can include a preparation that does not include botulinum toxin,for use as a diluents, control, blank sample, and/or zero standard. Inan alternative embodiment, such a kit can include a supply of media (forexample, a sensitivity enhancing media without botulinum toxin) and asupply of botulinum toxin stock. Such a supply of sensitivity enhancingmedia can be provided as a bulk supply or, preferably, as a series ofaliquots in individual containers containing characterized volumes. Itshould be appreciated that in such an embodiment the components can beprovided as separate components in order to accommodate differentstabilizing conditions (for example, different temperatures, bufferconditions, stabilizing reagents, etc.). In such an embodiment,directions are supplied to a user for addition of the botulinum toxinstock to the incomplete preparations in order to generate completepreparations that can be used as described above.

EXAMPLES

Temperature Effects.

Cell based assays to detect botulinum toxin (BOCELL™ assay) wereperformed at 35.0° C., 37.0° C., and 39.0° C. (Trials 1 and 2), and at37° C., 39° C., and 41° C. (Trial 3) using botulinum toxin/A holotoxinat concentrations ranging between 10⁻¹⁵ M to 10⁻⁹ M. The transfectedcells were exposed to one of the three temperatures while being exposedto the botulinum toxin. Dose/response curves were generated bycharacterizing emission ratios (YFP/CFP) at each concentration andplotting them as a function of botulinum toxin/A concentration. As shownin FIG. 1A, by increasing the temperature used in the assay from 37° C.to 39.0° C. or 41° C. the sensitivity to botulinum toxin (measured asEC₅₀ value) is enhanced more than 5 fold.

In the studies depicted in FIG. 1B the transfected cells were treatedwith either botulinum toxin/A holotoxin or botulinum toxin/A light chainand incubated at either 37.0° C. or 39.0° C. Botulinum toxin/A lightchain retains the ability to cleave the detection construct expressed bythe cells, but lacks the ability to bind to the specific cell surfacereceptor utilized by the intact holotoxin. In these studies botulinumtoxin/A light chain does not cleave the cleavage site containing portionof the reporting construct, even at high concentrations. This indicatesthat intact BoNT/A undergoes receptor-mediated toxin uptake process andactivation within the cell at both 37.0° C. and 39.0° C., and that theenhanced sensitivity is a receptor-mediated process.

Confirmation of this is found in the studies shown in FIG. 1C.Transfected cells were pre-treated with botulinum toxin/A heavy chain orthe equivalent vehicle prior to addition of botulinum toxin/A holotoxin.Botulinum toxin/A heavy chain lacks toxicity (i.e. proteolytic activity)and cannot cleave the detecting construct expressed by the cell, butbinds to an occupies the specific receptor bound by the holotoxin.Preincubation of the transfected cells with the heavy chain, whichcomprises the receptor binding domain, botulinum toxin/A holotoxinuptake and reporter cleavage at both 37.0° C. and 39.0° C., indicating arequirement for receptor-mediated endocytosis of BoNT/A holotoxin forreporter cleavage at elevated temperatures.

The effects of pre-treatment of cells using elevated temperatures isshown in FIG. 2. Cell based assays to detect BoNT (BoCell™ assay) wereperformed at 35.0° C., 37.0° C., and 39.0° C., using botulinum toxin/Aholotoxin at concentrations ranging between 10⁻¹⁵ M to 10⁻⁹ M. Thetransfected cells were exposed to one of these three temperatures beforebeing exposed to botulinum toxin, then exposed to the same or adifferent temperature among the three temperatures during exposure. Thesensitivity to botulinum toxin, characterized as a reduced EC50 value,was enhanced in transfected cells exposed to botulinum toxin at 39.0° C.Sensitivity to botulinum toxin at 39.0° C. was at least 3 fold greaterthan the sensitivity to botulinum toxin at 37.0° C., and at least morethan 10 fold compared to sensitivity to Botulinum toxin at 35.0° C.

The effects of elevated temperature combined with reduced osmolarity areshown in FIGS. 3A and 3B. Cell based assays to detect botulinum toxin(BOCELL™ assay) were performed at 35.0° C., 37.0° C., and 39.0° C.,where the transfected cells were exposed to Botulinum toxin in a cellmedia with an osmolarity of approximately 270 mOsm (i.e. normalosmolarity). Consistent with previous observations and as shown in FIG.3A, the observed sensitivity to botulinum toxin at 39.0° C. is increasedup to approximately 2 fold compared to sensitivity at 37.0° C. Similarstudies were performed using an otherwise identical cell culture mediawith an osmolarity of less than 250 mOSm. The results are shown in FIG.3B. Sensitivity to botulinum toxin at 39.0° C. is increased up toapproximately 7 fold compared to sensitivity to botulinum toxin at 37.0°C. Surprisingly, reduced osmolarity had relatively little effect at 37°C. and actually decreased sensitivity at 35° C., indicating asynergistic interaction between reduced osmolarity and elevatedtemperature.

As shown in FIG. 4A, the sensitivity enhancing effect of elevatedtemperature occurs within a narrow range of temperatures. Fluorescencedata from the cell-based assays performed at elevated temperatures showa loss of signal from a fluorescent protein of the construct at 41.0° C.when compared to lower temperatures, which can be indicative of poorcell health. Images of transfected cells under brightfield andfluorescence microscopy confirm poor cell health at 41.0° C., as shownin FIG. 4B. The transfected cells show poor morphology at 41.0° C.(brightfield) and an overall decrease and diffusion of the reporterprotein of the construct (YFP) at 41.0° C.

Selectivity of the temperature effect is shown in FIG. 5. Thetemperature studies shown above depict the results from using BoNT/A andtransformed cells expressing a construct that can be cleaved by BoNT/A.BoNT/A and BoNT/E both cleave sites within SNAP-25, and a reportingconstruct incorporating SNAP-25 or a portion of SNAP-25 that includesthese cleavage sites can potentially be used in the detection of eitherBoNT/A and BoNT/E. FIG. 5 shows the results BoNT/E cell-based assaysutilizing the BOCELL™ cells described above. Surprisingly, despiteutilizing the same cells and cell culture media, the use of elevatedtemperature within the range found to be effective for enhancement ofBoNT/A assay sensitivity (i.e. 39° C.) resulted in a decrease insensitivity for BoNT/E (shown as an elevated EC50 value). This indicatesthat the temperature effect may be selective for specific BoNTs.

Sodium Ion Effects.

Results of studies showing the effect of reduced sodium chloride (NaCl)concentration are shown in FIG. 6A. A custom basal cell culture mediawas prepared that contained no added NaCl. Variations of this custombasal media were prepared by adding NaCl at various concentrations andcell-based assays for botulinum toxin/A were performed using BOCELLcells. Cells were incubated for 3 hours prior to the application ofmedia containing BoNT/A at the indicated concentrations. Fluorescence ofthe fluorophores (i.e. YFP and CFP) of the construct expressed by thecells was characterized 48 hours after contacting the cells with BoNT/A.The highest concentration of NaCl (48 mM) represents the NaCl content ofthe conventional basal cell culture media. As shown, reduction of theNaCl concentration produces a dramatic enhancement of sensitivity(indicated by reduced EC50 values), finally resulting in a nearly50-fold increase in sensitivity in the absence of added NaCl.

The effects of reduced NaCl concentration on cell morphology(brightfield) in the absence of BoNT/A and the distribution of theconstruct within the transformed cells (YFP) in the absence and presenceof BoNT/A after 48 hours are shown in FIG. 6B. There is no evidence ofchanges in morphology or distribution of the construct at various NaClconcentrations in the cell culture media.

The impact of varying NaCl content of the cell culture media is shown inFIG. 7. A custom basal cell culture media was prepared that contained noadded NaCl. Variations of this custom basal media were prepared byadding NaCl at various concentrations and cell-based assays forbotulinum toxin/A were performed using BOCELL cells. Basal mediacontaining 48.3 mM NaCl represents the NaCl concentration of theconventional basal media. Cells were incubated with media containingBoNT/A at the indicated concentrations for 48, 72, and 96 hours.Fluorescence of the fluorophores (i.e. YFP and CFP) of the constructexpressed by the cells was characterized 48 hours after contacting thecells with BoNT/A. The highest concentration of NaCl (48 mM) representsthe conventional NaCl content of the basal cell culture media. As shown,the concentration of NaCl has little effect on the timing of thecell-based assay.

FIGS. 8A, 8B, and 8C show typical results of studies of the effects ofthe timing of the introduction of low sodium content media on thesensitivity of cell-based BoNT assays. FIG. 8A shows the results ofcells carrying appropriate reporting constructs incubated in a basalmedia with conventional sodium content prior to exposure (i.e.pre-incubation) to a concentration of BoNT/A in low sodium content basalmedia for either 4 hours or 24 hours. Following these time periods thecells were transferred to basal media with conventional sodium contentthat contained a corresponding concentration of BoNT/A, such that thetotal time spent exposed to BoNT/A was 48 hours. Cells were also exposedto BoNT/A in low sodium content basal media for the entire 48 hourperiod to provide control conditions. FIG. 8B shows typical results forsimilar studies performed using the low sodium content basal media forpre-incubation. It should be appreciated that the media used forpre-incubation of the cells had no discernible effect on the cells,indicating that pre-conditioning of the cells using low sodium contentmedia is not necessary.

The effect of pre-incubation was also examined in the studies shown inFIG. 8C, which shows typical results. Cells were pre-incubated with asupplemented conventional sodium content media (Media A) or with a lowsodium content custom media (Media B). Cells were then washed brieflywith either an unsupplemented conventional sodium content media (MediaC) or the low sodium content custom media prior to contact with BoNT. Asshown, pre-incubation in low sodium content media is not necessary togenerate the enhanced BoNT sensitivity.

Counterion Effects.

The sodium content of the cell culture media used in a cell-based BoNTassay of the inventive concept can be manipulated by adjusting theconcentration of sodium salts other than NaCl. As shown in FIG. 9A, areduction in the sodium bicarbonate (NaHCO₃) content of a basal media isalso effective at increasing the sensitivity of a cell-based BoNT assay.As shown in FIG. 9B, just as with NaCl large improvements in sensitivityare observed over relatively small changes in sodium content.

Ionic Strength and Osmolarity Effects.

The effects of removal of sodium from the media used in a cell-basedBoNT assay are not due to changes in ionic strength. FIG. 10 showstypical results from studies in which a series of custom media havingconventional (i.e. 70%) and reduced (i.e. 25%) sodium content and inwhich sodium is replaced by potassium at the same concentrations. Whilethe enhancement of sensitivity in a cell-based BoNT assay is evident onreduction of sodium concentration, a similar enhancement is not observedwhen sodium is replaced with potassium and the concentrationsubsequently reduced. As such the effect is independent of ionicstrength and can be seen as ion-specific and/or ion-selective.

FIG. 11 shows the results of supplementing low sodium content media withnonionic substances to increase ionic strength. Cell based BoNT assayswere performed in culture media containing 48 mM NaCl (70% Neurobasal,total [Na+]=53 mM), 0 mM NaCl (70% custom 0 mM NaCl, total [Na+]=19 mM),and 0 mM NaCl media supplemented with either sucrose or trimethylamineN-oxide (TMAO). Both sucrose and TMAO are commonly used to adjustosmolarity. The sensitivity enhancement produced by the reduction ofsodium in the culture media remains despite adjusting the osmolarity tothe equivalent of 48 mM NaCl. The effects of reduction in sodium contentin the culture media utilized in cell-based BoNT assays is thereforeindependent of osmolarity.

Mechanistic Studies of Low Sodium Content Media.

There are a variety of mechanisms that may be involved in enhancement ofthe sensitivity of cell-based BoNT assays through the use of low sodiumcontent culture media. FIG. 12A shows typical results obtained instudies directed towards blocking cell surface receptor-mediated uptakeof BoNT by cells in low sodium content culture media. Such cells weretreated with a recombinant heavy chain fragment of BoNT/A (HcR/A, at 1μM) prior to exposure of the cells to the intact BoNT/A holotoxin. Suchheavy chain fragments of BoNT/A bind to the same cell surface receptorsas the holotoxin but lack proteolytic activity and cannot cleave thedetecting construct. As shown, blocking these receptor sites effectivelyblocks the toxic effects of the BoNT/A holotoxin when applied in lowsodium content culture media at all but high holotoxin concentrations.

The recombinant light chain fragment of BoNT/A (Lc/A) retains theproteolytic activity of the BoNT/A holotoxin, but lacks the ability tobind to the cell surface receptors utilized for internalization of theholotoxin. FIG. 12B shows typical results for a study on theinternalization of the BoNT/A light chain by cells in low sodium contentculture media. As shown Lc/A has minimal impact on these cells,indicating that nonspecific endocytosis is not a primary factor in thesensitivity enhancement seen with low sodium content media.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to a firstand a second steps, the text should be interpreted to mean that thefirst and second steps can be practiced in any order, not that the claimrequires both element should be present or two elements are in suchorder. Where the specification claims refers to at least one ofsomething selected from the group consisting of A, B, C . . . and N, thetext should be interpreted as requiring only one element from the group,not A plus N, or B plus N, etc. Similarly, the inventive subject matteris considered to include all possible combinations of the disclosedelements. Thus if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

What is claimed is:
 1. A method of increasing sensitivity of a cellbased assay for Botulinum neurotoxin, comprising: (i) providing, in amedia having a sub-physiological osmolarity and a temperature of atleast 39° C., a transfected cell comprising a reporting peptidecleavable by Botulinum neurotoxin; (ii) contacting the cell withBotulinum neurotoxin; and (iv) measuring a cleavage product derived fromthe reporting peptide, thereby providing a synergistic decrease in EC₅₀for Botulinum neurotoxin relative to the cell-based assay performed atphysiological temperature and physiological osmolarity.
 2. The method ofclaim 1, wherein the reporting peptide comprises: a terminus comprisinga reporter-containing portion, wherein the reporter-containing portionexhibits a signal; and, a cleavage site that interacts with theBotulinum neurotoxin in a manner that produces a cleavage of thereporter-containing portion from a remainder of the reporting peptide,wherein measuring is performed by monitoring changes in the signal. 3.The method of claim 1, wherein the media has an osmolarity of at least250 mOsm.
 4. The method of claim 1, wherein the EC₅₀ for Botulinumneurotoxin is decreased by at least a factor of three relative to thecell-based assay performed at physiological temperature and osmolarity.5. The method of claim 1, wherein the Botulinum neurotoxin is arecombinant toxin.
 6. The method of claim 4, wherein the recombinanttoxin is directed to a non-neuronal cell.
 7. The method of claim 1,wherein the transfected cell is a non-neuronal cell.